1. Calmodulin and Phosphorylase Interaction By James Proestos Biochemistry and Biophysics Department Dr. Sonia Anderson’s Lab Agriculture and Life Science Building

2. Calcium Activator of many cellular processes (cell signaling) –Triggers muscle proteins to contract –Activates many enzymes

3. Calmodulin Information Is found in all animal and plant tissues Binding of calcium controls its ability to bind to a protein to regulate the target protein’s activity.

4. Calmodulin Structure

5. Target Calmodulin Ca

6. Bound Calmodulin Ca

7. Bound Calmodulin Ca Target Protein

8. Bound Calmodulin Ca Bound Protein

9. Glycogen Phosphorylase Information Found in fast twitch muscle tissue It catalyzes the breakdown of glycogen Controlled by phosphorylation/dephosphorylation

10. The Phosphorylated and Unphosphorylated States of Glycogen Phosphorylase B Serine Glucose A

11. Cascade of Reactions in Glycogen Degradation

12. The Interaction of Proteins in Glycogen Cascade Phosphorylase Kinase becomes active by calcium binding to the intrinsic calmodulin The phosphorylase kinase interacts with the glycogen phosphorylase It is not known if the calmodulin can readily bind with glycogen phosphorylase in this interaction

13. Phosphorylase binds to calmodulin Hypothesis Malencik and Anderson proposed that calmodulin binding regions are often sites of regulation by serine- threonine phosphorylation/dephosphorylation

14. Phosphorylase binds to calmodulin Hypothesis Malencik and Anderson proposed that calmodulin binding regions are often sites of regulation by serine- threonine phosphorylation/dephosphorylation Question Is the calmodulin binding region of phosphorylase b the same as the phosphorylation site and how does phosphorylation affect this binding to calmodulin?

15. Purification of Phosphorylase B Grind rabbit muscle Spin in a centrifuge Remove the pellet Ammonium sulfate precipitation and crystallize Repeat crystallization several times

16. Phosphorylase Purification Scan of phosphorylase gel 96 K 68 K 42 K 29 K 18 K 12 K

17. Phosphorylase Purification

18. Scan of concentrated protein

19. Purification of Calmodulin Grind bovine brain Spin in centrifuge and remove pellet Pass the supernatant through several columns

20. Purification of Calmodulin

21. Scan of calmodulin gel 96 K 42 K 29 K 18 K 12 K

22. Experimental Plan Cleave the glycogen phosphorylase protein into peptides Isolate peptides of interest by conventional column chromatography Determine the binding of the peptides using a calmodulin affinity column Identify the peptide(s) from the calmodulin affinity column by mass spectrometry and/or amino acid analysis Phosphorylate the peptide(s) and compare its affinity for calmodulin to that of the unphosphorylated peptide (by fluorescence).

23. Cleavage of Phosphorylase B 184114 CNBR RXN HydroxylamineSubtilisin 114264 265841 114134 135259 260497 498841 1 242 442 91 350 604 14 351428

24. Cleavage of Phosphorylase B Cyanogen bromide is our tool to cleave at methionine residues The resulting peptides will be the focus of the binding of calmodulin

25. Cleavage of Phosphorylase B Num From-To MW pI 1 1- 91 11054.63 10.17 Residue that will be observed, serine-14 is site of phosphorylation 2 92- 99 921.07 11.15 3 100-119 2184.45 2.74 4 120-147 2940.24 3.35 5 148-176 3229.75 8.80 6 177-224 5662.32 4.95 7 225-241 1938.17 6.81 8 242-350 12565.35 8.51 9 351-428 9322.85 7.36 10 429-441 1446.71 7.02 11 442-604 19129.32 9.64 12 605-615 1102.30 9.80 13 616-618 349.47 10.20 14 619-679 6480.42 4.59 15 680-682 425.57 9.75 16 683-692 991.20 6.96 17 693-699 735.79 2.91 18 700-713 1591.74 2.87 19 714-764 6134.86 4.45 20 765-766 263.38 6.96 21 767-800 4333.90 8.61 22 801-840 4545.12 10.20

26. Separation of Glycogen Phosphorylase Peptides Gel filtration –Separation based on size Cation exchange –Separation based on charge (pI >7) High Performance Liquid Chromatography –Separation based on hydrophobicity Analyze peptides –Peptide 1-91 –Synthetic peptide 6-25 –Calmodulin binding peptide

27. Cleavage of Phosphorylase B 184114 CNBR RXN 1 242 442 91 350 604 14 351428

28. Gel Filtration Peptide Fragments

29. Cation Exchange

30. Amino Acid Analysis of Glycogen Phosphorylase

31. Synthesized Peptide 1 91 14 DQEKRKQI S VRGLAGVENVT MW= 2228 MW= 11054.63

32. HPLC Purification of Peptide 6-25

33. Mass Spectrometry of Peptide 6-25

34. Calmodulin/Phosphorylase B Interaction bound calmodulin gel peptides that do not bind to calmodulin Phosphorylase peptides

35. Determine affinity of calmodulin-peptide complex Phosphorylate peptides and recheck affinity Isolated peptides A)Peptide(1-91) B)Peptide(6-25) C)CaM Binding Peptide Analysis of Calmodulin/Glycogen Phosphorylase Interaction

36. Analysis of Phosphorylation Interaction Peptide-P+P Intact phosphorylase+++ND CNBr digest (mixture)+++ + Peptide 351-408 + + Peptide 1-91 ++++ ++ Synthetic peptide (6-25)none none Glutathione (control) none none

37. Future Work Determine and verify phosphorylation and stoichiometry of the various peptides by the use of ATP32 Determine the affinity of the peptides quantitatively by the use of fluorescence titration Complete sequence and/or mass spectroscopy information on the petide/s that bind to the solid support calmodulin column and, if enough material is isolated, to perform calmodulin binding Redetermine the binding of peptide 6-25 utilizing different approaches, if necessary, to verify that it does not bind to calmodulin Subfragment peptide 1-91 and redetermine its binding to calmodulin

38. Acknowledgements Howard Hughes Medical Institute Dr. Sonia Anderson Dean Malencik Department of Biochemistry and Biophysics Kevin Ahern

39. Cleavage of Phosphorylase B